The Rh system is a highly polymorphic blood group system that plays an important role in haemolytic transfusion reactions, neonatal haemolytic disease and autoimmune haemolytic anemia. There are two different, but highly homologous, genes in the Rh system. One gene (RhD) encodes the D polypeptide, while the other gene (RHCE) encodes the CcEe polypeptide. RhD carries the D antigen—the most potent blood group immunogen. This antigen is absent from a relatively large segment (15-17%) of the population (the Rh-negative phenotype), as a result of RhD gene deletion or other RhD gene alterations (e.g., gene conversion, Pseudogene RhD psi). As used herein the term “psi” refers to the Greek symbol “ψ.” RHCE exists in four allelic forms and each allele determines the expression of two antigens in Ce, ce, cE or CE combination (RHCE is the collective name of the four alleles).
Tests for determining RhD type are critical for a wide range of applications. When blood of a rhesus D (RhD) positive donor is given to an RhD negative patient there is a high chance that alloantibody formation occurs. RhD antibodies will lead to rapid destruction of RhD-positive red cells and to transfusion reactions. Furthermore, when a woman with red cell or platelet antibodies becomes pregnant, those antibodies can cross the placenta and can destruct the red cells or the platelets of the unborn child.
In the past, nucleic acid-based RhD typing was performed on fetal nucleic acid procured through invasive means. However, conventional invasive sampling techniques that analyze fetal DNA from amniotic fluid or chorionic villus are costly and may lead to miscarriage and sensitization of the mother. An alternative source of fetal DNA was shown to be maternal plasma and serum (Lo et al., Lancet 350, 485-487 (1997)).